AP Biology Lab 2: Enzyme Catalysis

Background: Enzymes are the catalysts of biological systems. They speed up chemical reactions in biological systems by lowering the activation energy, the energy needed for molecules to begin reacting with each other. Enzymes do this by forming an enzyme-substrate complex that reduces energy required for the specific reaction to occur. Enzymes have specific shapes and structures that determine their functions. The enzyme’s active site is very selective, allowing only certain substances to bind. If the shape of an enzyme is changed in any way, or the protein denatured, then the binding site also changes, thus disrupting enzymatic functions. Enzymes are fundamental to the survival of any living system and are organized into a number of groups depending on their specific activities. Two common groups are catabolic enzymes (“cata” or “kata-” from the Greek “to break down”) — for instance, amylase breaks complex starches into simple sugars — and anabolic enzymes (“a-” or “an-” from the Greek “to build up”). (You may know this second word already from stories about athletes who have been caught using anabolic steroids to build muscle.) Catalytic enzymes, called proteases, break down proteins and are found in many organisms; one example is bromelain, which comes from pineapple and can break down gelatin. Bromelain often is an ingredient in commercial meat marinades. Papain is an enzyme that comes from papaya and is used in some teeth whiteners to break down the bacterial film on teeth. People who are lactose intolerant cannot digest milk sugar (lactose); however, they can take supplements containing lactase, the enzyme they are missing. All of these enzymes hydrolyze large, complex molecules into their simpler
components; bromelain and papain break proteins down to amino acids, while lactase breaks lactose down to simpler sugars. Anabolic enzymes are equally vital to all living systems. One example is ATP synthase, the enzyme that stores cellular energy in ATP by combining ADP and phosphate. Another example is rubisco, an enzyme involved in the anabolic reactions of building sugar molecules in the Calvin cycle of photosynthesis.

Pre-Lab: Define the following enzyme vocabulary words...enzyme, catalyst, substrate, product, active site

Exercise 2A: Test of Catalase Activity (Done as a demo for class)

Drawing of demo for 2A:

Analysis Questions for 2A:
1. What is the enzyme in the reaction?

2. What is the substrate in the reaction?

3. What is the product in the reaction?

4. How could you show that the gas evolved is O₂?

5. How does the reaction compare to the one using the unboiled catalase? Explain the reason for this difference.

6. What happened when potato/liver was added to hydrogen peroxide? What do you think would happen if the potato/liver had been boiled?

Exercise 2B: The Base Line Assay

Background:

In this experiment the disappearance of the substrate, H₂O₂, is measured as follows:

1) A enzyme is mixed with the substrate in a beaker. The enzyme catalyzes the conversion of H₂O₂ to H₂O and O₂(gas).

2) Before all of the H₂O₂ is converted to H₂O and O₂, the reaction is stopped by adding sulfuric acid (H₂SO₄). The H₂SO₄ lowers the pH, denatures the enzyme, and thereby stops the enzymes catalytic activity.

3) After the reaction is stopped, the amount of substrate (H₂O₂) remaining in the beaker is measured. To measure this quantity, potatssium permanganate is used. Potassium permanganate (KMnO₄) in the presence of H₂O₂ and H₂SO₄ reacts as follows:

5 H₂O₂+ 2 KMnO₄ + 3 H₂SO₄ --> K₂SO₄ + 2 MnSO₄ +8 H₂O+ 5 O₂

Note that H₂O₂ is a reactant for this reaction. Once all of the H₂O₂ has reacted, any more KMnO₄ added will be in excess and will not decomposed. The addition of excess KMnO₄ causes the solution to have a permanent pink or brown color. Therefore, the amount of H₂O₂ remaining is determined by adding KMnO₄ until the whole mixture stays a faint pink or brown permanently. Add no more KMnO₄  after this point. The amount of KMnO₄ added is a proportional measure of the amount of H₂O₂ remaining.

Drawing of Set Up for 2B:

Results for 2B:

Example Exercise 2D: An Enzyme-Catalyzed Rate of H₂O₂ Decomposition

Purpose Question: What is the effect of time enzyme is allowed to work on the amount of hydrogen peroxide left in the container? (Can make up your own question to test)

Hypothesis and Null Hypothesis:

Procedure:

Drawing of Set Up for 2D:

Results for 2D:
 

Analysis Questions for 2D:

1) Do calculations to determine Amount of H₂O₂ reacted in each test group by subtracting the amount of KMnO4 that you added to turn solution brown in each test group from the amount of KMnO4 added in your control group. (Baseline-Test Group= H2O2 that reacted)

2) Make a graph of your data. Be sure to label your axis correctly with the independent and dependant variables and appropriate title.

3) Using the formula, rate = amount of H2O2 that reacted/time you allowed enzyme to react, determine the rate of the reaction for each test group.

4) Discuss rates you determined. When is the rate highest? Explain why? When is the rate the lowest? For what reasons is the rate low?

5) Explain the inhibiting effect of sulfuric acid on the function of the enzyme. Relate this to enzyme structure and chemistry.

6) Determine Chi-Square Analysis for your Data (use Control Group or Baseline Assay as expected) or Standard Deviation and Standard Error if you were able to get data for multiple trials and put error bars on graph.

Conclusion:

As stated on blue lab sheet. If you calculated chi-square analysis, then make sure to use data to support which hypothesis you supported. If you calculated standard deviation make sure include data when you discuss errors. Make a reference to the fact that you only did one statistical analysis for which ever one you didn't do during the discussions.